STR GENOTYPING BY DIFFERENTIAL HYBRIDIZATION

摘要

In a method of deducing the number of repeat units in a selected short tandem repeat (STR) in a genomic sample, at least a single stranded target DNA generated from a genomic sample comprising a selected STR, an STR probe (P1,P1′), a reference probe (P2), and two blockers (B1,B2) are provided, and at least three differential hybridization experiments are carried out, based on which the number of STR probe oligonucleotides (P1,P1′) bound per target DNA strand in each differential hybridization experiment is determined. The method further comprises the step of comparing these numbers of STR probe oligonucleotides (P1,P1′) bound per target DNA strand in the differential hybridization experiments for deducing the number of repeat units in the selected STR on the single stranded target DNA strand. Also disclosed are kits for carrying out STR genotyping by differential hybridization.

主权项

1. A method of deducing the number of repeat units in a selected short tandem repeat (STR) in a genomic sample, the method comprising the steps of:
a) providing at least:
al) a single stranded target DNA generated from a genomic sample comprising a selected STR;a2) an STR probe (P1, P1′) with a first fluorescent label, the at least one STR probe (PI, PI′) being an oligonucleotide which comprises a sequence complementary to a defined number of repeat units of the selected STR on the single stranded target DNA;a3) a reference probe (P2) with a second fluorescent label, which is different from the first fluorescent label, the reference probe (P2) being an oligonucleotide which comprises a sequence complementary to a 5′- or a 3′-flanking sequence of the selected STR on the single stranded target DNA;a4) two blockers (B1,B2) which are oligonucleotides, wherein a first blocker (Bl) comprises a sequence complementary to a sequence of the 5′ flanking region of the STR and to at least one of the STR repeat units adjacent to that 5′ flanking region, and wherein a second blocker (B2) comprises a sequence complementary to a sequence of the 3′ flanking region of the STR and to at least one of the STR repeat units adjacent to that 3′ flanking region; b) carrying out at least the following three differential hybridization experiments by mixing in each experiment an amount of the single stranded target DNA with:
bl) the at least one STR probe (P1, P1′) and the reference probe (P2), and allowing hybridization to the single stranded target DNA in a first differential hybridization experiment;b2) the at least one STR probe (PI, PI′), the reference probe (P2) and one of the at least two blockers (B1,B2), and allowing hybridization to the single stranded target DNA in a second differential hybridization experiment; andb3) the at least one STR probe (PI, PI′), the reference probe (P2) and the two blockers (B1,B2), and allowing hybridization to the single stranded target DNA in a third differential hybridization experiment; c) measuring for each differential hybridization experiment the intensity of the fluorescence provided by the at least one STR probe (PI, PI′) bound to the repeat units of the selected STR; d) measuring for each differential hybridization experiment the intensity of the fluorescence provided by the reference probe (P2) bound to one of the flanking sequence of the single stranded target DNA; e) correlating for each differential hybridization experiment the fluorescence intensity of the at least one STR probe (PI, PI′) measured in step c) to the fluorescence intensity of the reference probe (P2) measured in step d), thereby determining for each differential hybridization experiment the number of STR probe oligonucleotides (P1, P1′) bound per target DNA strand, and f) comparing the numbers of STR probe oligonucleotides (PI, PI′) bound per target DNA strand determined in the differential hybridization experiments for deducing the number of repeat units in the selected STR on the single stranded target DNA strand.